Expression of selected genes involved in steroidogenesis in the course of enucleation-induced rat adrenal regeneration

Abstract

The enucleation-induced (EI) rapid proliferation of adrenocortical cells is followed by their differentiation, the degree of which may be characterized by the expression of genes directly and indirectly involved in steroid hormone biosynthesis. In this study, out of 30,000 transcripts of genes identified by means of Affymetrix Rat Gene 1.1 ST Array, we aimed to select genes (either up- or downregulated) involved in steroidogenesis in the course of enucleation-induced adrenal regeneration. On day 1, we found 32 genes with altered expression levels, 15 were upregulated and 17 were downregulated [i.e., 3β-hydroxysteroid dehydrogenase (Hsd3β), nuclear receptor subfamily 0, group B, member 1 (Nr0b1), cytochrome P450 aldosterone synthase (Cyp11b2) and sterol O-acyltransferase 1 (Soat1)]. On day 15, the expression of only 2 genes was increased and that of 3 was decreased. The investigated genes were clustered according to an hierarchical clustering algorithm and 4 clusters were obtained. Quantitative PCR (qPCR) confirmed the much lower mRNA expression levels of steroidogenic acute regulatory protein (StAR) during the regeneration process compared to the control, while the cholesterol side-chain cleavage enzyme (cholesterol desmolase; Cyp11a1) and Hsd3β genes presented similar expression profiles throughout the entire regeneration process. Cyp11b2 mRNA levels remained very low during the whole regeneration period. Fatty acid binding protein 6 (Fabp6) was markedly upregulated, whereas hormone-sensitive lipase (Lipe) was downregulated. The expression of Soat1 was lowest on regeneration day 1 and, subsequently, its expression increased from there on, reaching levels higher than the control. Dosage-sensitive sex reversal, adrenal hypoplasia critical region, on chromosome X, gene 1 (Dax-1) mRNA levels were lowest on day 1 of the experiment; however, throughout the entire experimental period, there were no statistically significant differences observed. After the initial decrease in steroidogenic factor 1 (Sf-1) mRNA levels observed on the 1st day of the experiment, a marked upregulation in its expression was observed from there on. Data from the current study strongly suggest the role of Fabp6, Lipe and Soat1 in supplying substrates of regenerating adrenocortical cells for steroid synthesis. Our results indicate that during the first days of adrenal regeneration, intense synthesis of cholesterol may occur, which is then followed by its conversion into cholesteryl esters. Moreover, our data demonstrated that in enucleation-induced regeneration, the restoration of genes involved in glucocorticoid synthesis is notably shorter than that of those involved in aldosterone synthesis.

Visinin-like peptide 1 in adrenal gland of the rat. Gene expression and its hormonal control

“…Expression of Cyp11b2 gene serves as a marker of the adrenal ZG. Following enucleation, by in situ hybridization, transcriptome analysis and QPCR, a marked decrease in Cyp11b2 gene expression levels were observed between days 2 and 20 of experiment [10,11,56]. As demonstrated in present study, similar pattern of expression was found for Vsnl1 and expression levels of both genes were positively correlated (almost strong positive correlation r = 0.68).…”

“…The applied methods were described earlier [1,2,[45][46][47][53][54][55][56]. From collected cells, samples of adrenal zones and from samples of entire adrenal glands, total RNA was extracted using TRI Reagent (Sigma) and, then, purified on columns (Rnasy Mini Kit, Qiagen).…”

“…Microarray expression analysis was also described earlier [54][55][56]. RNA subjected to microarray studies was obtained from the above described experiments.…”

“…Using the above mentioned primers, SYBR green detection system was applied as described earlier [45][46][47][54][55][56]. Every of 20 l reaction mixtures contained 4 l template cDNA (standard or control), 0.5 M of specific primer and a previously determined optimum MgCl 2 concentration (3.5 M for one reaction).…”

Effect of ACTH and hCG on the Expression of Gonadotropin-Inducible Ovarian Transcription Factor 1 (Giot1) Gene in the Rat Adrenal Gland

“…Using the primers mentioned above, SYBR green detection system was applied as described earlier [56,57,61,62,63,64]. Every of 20 μL reaction mixtures contained 4 μL template cDNA, 0.5 µM of specific primer and a previously determined optimum MgCl 2 concentration (3.5 M for one reaction).…”