engleski

Expression profiling of cholesterol homeostasis genes by cDNA microarrays

Cholesterol is an essential component of eukaryotic membranes. Its biosynthesis takes place in all cell types, primarily in the liver, colon, adrenal, brain and sex glands. Cholesterol metabolites (steroid hormones, bile acids, vitamin D and oxysterols) have many different functions. Due to these different roles, biosynthesis of cholesterol and of its metabolites must be regulated by different factors and signalling pathways in a tissue-specific manner. Several studies concerning regulation of individual genes by either the sterol-dependent pathway (negative cholesterol feedback loop), cAMP-dependent pathway or by nuclear receptors are available. However, a global approach which would enable understanding the reponse of the entire set of cholesterol homeostatic genes to tissue-specific endogenous and exogenous stimuli, is lacking at present. We have designed a limited human and mouse cholesterol homeostasis cDNA microarray, to study expression of genes, encoding enzymes involved in cholesterol biosynthesis, esterification, metabolism and regulation. The influence of various physiological conditions, such as the level of cholesterol (negative cholesterol feedback loop), cAMP-dependent stimuli, xenobiotic-mediated activation by nuclear receptors, on the transcriptome of cholesterol homeostasis is being evaluated. This approach will be applied also to get a clearer picture of CYP51 (lanosterol 14?-demethylase) in mammalian reproduction. CYP51 is a cytochrome P450 enzyme that participates in the postsqualene portion of cholesterol biosynthesis. Enzymes with CYP51 activity are found in three eukaryotic phyla and in prokaryotes. CYP51 metabolic product MAS (meiosis activating sterol), is belived to have an important role in mammalian reproduction. We propose the existance of a special regulatory pathway involved in production of MAS in male germ cells. Several experiments with by stimuli treated immortal cell lines of human (HepG2, JEG3) and mouse origin as well with mouse tissues (liver, brain, testis) have been performed and used for optimization of the method. Experiments show a low expression level of most cholesterol homeostasis genes in all immortal cell lines. There is a marked difference in the pattern of expressed genes in tissues compared to from tissues-derived immortal cells. Computation of a large set of data is in progress and results will be presented on the poster.

expression profiling; cholesterol homeostasis genes; cDNA microarray

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