原田 信広

Our in vitro characterization showed that physiological concentrations of estrogen partially suppressed the I-Kr channel current in guinea pig ventricular myocytes and the human ether-a-go-go-related gene (hERG) channel currents in CHO-K1 cells regardless of estrogen receptor signaling and revealed that the partially suppressed hERG currents enhanced the sensitivity to the hERG blocker E-4031. To obtain in vivo proof-of-concept data to support the effects of estrogen on cardiac electrophysiology, we here employed an aromatase knockout mouse as an in vivo estrogen-null model and compared the acute effects of E-4031 on cardiac electrophysiological parameters with those in wild-type mice (C57/BL6J) by recording surface electrocardiogram (ECG). The ablation of circulating estrogens blunted the effects of E-4031 on heart rate and QT interval in mice under a denervation condition. Our result provides in vivo proof of principle and demonstrates that endogenous estrogens increase the sensitivity of E-4031 to cardiac electrophysiology.

The human CYP19 gene encodes aromatase, which converts androgens to estrogens. CYP19 mRNA variants are transcribed mainly from three promoters. Quantitative RT-PCR was used to measure the relative amounts of each of the three transcripts and determine the on/off state of the promoters. While some of the promoters were silent, CYP19 mRNA production differed among the other promoters, whose estimated transcription levels were 0.001% to 0.1% of that of the TUBB control gene. To investigate the structural aspects of chromatin that were responsible for this wide range of activity of the CYP19 promoters, we used a fractionation protocol, designated SEVENS, which sequentially separates densely packed nucleosomes from dispersed nucleosomes. The fractional distribution of each inactive promoter showed a similar pattern to that of the repressed reference loci; the inactive regions were distributed toward lower fractions, in which closed chromatin comprising packed nucleosomes was enriched. In contrast, active CYP19 promoters were raised toward upper fractions, including dispersed nucleosomes in open chromatin. Importantly, these active promoters were moderately enriched in the upper fractions as compared to active reference loci, such as the TUBB promoter; the proportion of open chromatin appeared to be positively correlated to the promoter strength. These results, together with ectopic transcription accompanied by an increase in the proportion of open chromatin in cells treated with an H3K27me inhibitor, indicate that CYP19 mRNA could be transcribed from a promoter in which chromatin is shifted toward an open state in the equilibrium between closed and open chromatin.

The post-translational regulation of aromatase has not been well characterized as compared with transcriptional regulation. Several studies of post-translational regulation have focused on decreases in catalytic activity following phosphorylation. We report here dual post-translational regulation of aromatase, at the catalytic activity and protein levels. Microsomal aromatase prepared from JEG-3 cells was rapidly inactivated and subsequently degraded in the presence of a cytosolic fraction with calcium, magnesium, and ATP. In a reconstituted system consisting of microsomal and cytosolic fractions, aromatase was protected from protein degradation by treatment with alkaline phosphatase, whereas degradation was enhanced by treatment with calcineurin inhibitors (FK506 and cyclosporin A). Furthermore, aromatase was protected from degradation by treatment with kinase inhibitors, especially the calcium/calmodulin kinase inhibitors KN62 and KN93. Similarly to the reconstituted system, aromatase in cultured JEG-3 cells was protected from degradation by KN93, whereas FK503 increased degradation in the presence of cycloheximide, although cellular aromatase mRNA levels were unchanged by these reagents. Knockdown of calcineurin and calcium/calmodulin kinase II (CaMKII) with small interfering RNAs resulted in a dose-dependent increase in aromatase degradation and protection from degradation, respectively. The cytosol fraction-dependent phosphorylation of microsomal aromatase was inhibited by calcineurin, KN62, and KN93, and promoted by CaMKII and FK506. These results indicate that aromatase is regulated acutely at the catalytic activity level and subsequently at the enzyme content level by CaMKII/calcineurin-dependent phosphorylation/dephosphorylation.