Abstract 1260: Novel Mechanism of Aldosterone Synthesis: Critical Role of the Transcriptional Repressor NRSF System in Aldosterone Synthesis in Human Adrenocortical Cells (H295R)
Background and Aim: Aldosterone plays important roles in pathogensis of chronic heart failure. Aldosterone is synthesized by a specific synthase, CYP11B2, of which expression is regulated by Ca2+ influx through T type calcium channel (mainly Cav3.2 in adrenal) in response to angiotensinII (AngII) and extracellular K+. However, molecular mechanism of aldosterone synthesis is not fully understood. Recently we found that the inhibition of neuron restrictive silencer factor (NRSF), which binds to neuron restrictive silencer element (NRSE) and suppresses NRSE-containing genes, is involved in re-induction of many cardiac embryonic genes. Computer analysis revealed that NRSE sequence is located in intron8–9~exon9 of the CYP11B2 gene and the first intron of the CACNA1H gene that encodes α subunit of Cav3.2. The aim of the present study is to elucidate the roles of the NRSE/NRSF system on aldosterone synthesis in human adrenocortical cells (H295R).
Methods and Result: Inhibition of endogenous NRSF function by dominant-negative NRSF adenovirus (AD/dnNRSF) markedly increased aldosterone secretion in association with 26.9-fold increase of CYP11B2 mRNA. AD/dnNRSF also augmented CACNA1H mRNA to 330 % of control level. In the absence of AD/dnNRSF, AngII increased CYP11B2 mRNA by 18.3-fold, but in the presense of overexpressed AD/dnNRSF AngII increased it by only 1.5-fold. Next, to further confirm the NRSF-dependent transcriptional regulation of the CYP11B2 gene, we subcloned NRSE or mutated NRSE (mtNRSE) sequence into downstream of a luciferase gene driven by CYP11B2 promoter. Co-transfection of dnNRSF with these reporter genes showed 4.4-fold increase in the luciferase activity of CYP11B2/luc/NRSE. Moreover, co-transfection also did show 2.4-fold increase of the luciferase activity of CYP11B2/luc/mtNRSE, suggesting the involvement of inditrect effect of another NRSE-containing gene, CACNA1H. The blocker of Cav3.2, effonidipine, completely inhibited the dnNRSF-inducible luciferase activity of CYP11B2/luc/mtNRSE.
Conclusion: These findings suggest that NRSE/NRSF system is involved in basal and AngII-induced expression of CYP11B2. This system plays a key role in the aldosterone synthesis through gene expression of CYP11B2 and CACNA1H in H295R.