Since free testosterone diffuses into a
Since free testosterone diffuses into a target organ , a remarkable increase in free testosterone in plasma is expected to enhance androgen response in target organs including the prostate. In fact, the results of the present study showed testosterone and DHT levels, and mRNA expression levels of all target genes of the AR examined in this study were significantly increased in the prostates of Cyp3a−/− mice (Figs. 2 C and 3 A). In addition, protein expression levels of SCAP were increased in the prostates of Cyp3a−/− mice (Fig. 3B). The results of ChIP assays also showed that specific binding of the AR to the ARE-containing region of the Sbp and Prkcd promoter ,  was significantly more abundant in the prostates of Cyp3a−/− mice than in the prostates of WT mice (Fig. 3C). Furthermore, treatment with bicalutamide to Cyp3a−/− mice decreased the mRNA expression levels of AR target genes in the prostates (Fig. 5A). The fold decrease caused by bicalutamide were larger than the fold increase caused by Cyp3a deficiency, which implied bicalutamide diminished their physiological and induced expressions regulated by AR in the prostates of Cyp3a−/− mice. These findings suggest that androgen response via AR activation is stimulated in the prostate of Cyp3a−/− mice through the increases of prostatic testosterone levels. Activated AR has been reported to increase the expression of target genes of SREBP2 involved in cholesterol synthesis and transport through transactivation of SCAP, which enhances maturation of SREBP in LNCaP WY-14643 mg , . In accordance with the findings using LNCaP cells, the present study showed that protein expression levels of SCAP and mRNA expression levels of SREBP2 target genes involved in the synthesis and transport of cholesterol were increased in the prostates of Cyp3a−/− mice (Figs. 3 B and 4 B), and they were decreased by bicalutamide treatment to Cyp3a−/− mice (Fig. 5B). In addition, expression levels of mature SREBP2 were elevated in the prostates of Cyp3a−/− mice (Fig. 4A). Finally, total cholesterol levels were increased in the prostates of Cyp3a−/− mice (Fig. 4C). The findings suggest that Cyp3a deficiency increases testosterone in plasma and prostates, which enhances cholesterol synthesis via the SCAP-SREBP2 pathway in the prostate as reported previously in LNCaP cell , . Although high cellular cholesterol levels suppress activation of SREBP2 , it seemed that SCAP-mediated activation of SREBP2 exceeded the cholesterol-mediated suppression of SREBP2 in the prostates of Cyp3a−/− mice. In addition, it has been reported that transcription of Srd5a2 gene is enhanced by activated SREBP2 , suggesting that activated SREBP2 might increase mRNA expression levels of SRD5A2 in the prostates of Cyp3a−/− mice. In this context, we previously reported that SREBP2 was activated in the livers of Cyp3a−/− mice, possibly due to impaired synthesis of oxysterols (e.g., 25-hydroxycholesterol), which can suppress proteolytic activation of SREBP2 , . Therefore, the results of this study may imply that an increased level of free testosterone also causes the activation of SREBP2 in livers of Cyp3a−/− mice. Further study is needed to clarify the impact of AR activation on SREBP2 activation in the livers of Cyp3a−/− mice. The present study showed that Cyp3a deficiency dramatically increased free testosterone in plasma, which stimulated androgen response and enhanced cholesterol synthesis in the prostate. Zhang et al. reported that altered plasma level of testosterone affects androgen response including physiological development of prostate in castrated mice . However, we could not find any difference in the weight of prostate between Cyp3a−/− and WT mice (data not shown). In non-castrated mice, increased testosterone may not induce such physiological effects. On the other hand, this remains possible that increased testosterone and cholesterol levels affect the disease state of the prostate. For example, epidemiological evidence suggested that high levels of circulating testosterone is related to the risk of benign prostate hypertrophy and prostate cancer , . In addition, high-fat diets increased the risk of prostate cancer by accumulation of cholesterol , , whereas cholesterol-lowering drugs (e.g., statins) reduced the risk of advanced prostate cancer , . Thus, decreased activity of CYP3A may lead to deterioration of benign prostate hypertrophy and prostate cancer.