RESUMEN
Purkinje cell protein 4 (PCP4) is a calmodulin (CaM)-binding protein that accelerates calcium association and dissociation with CaM. It has been previously detected in aldosterone-producing adenomas (APA), but details on its expression and function in adrenocortical tissues have remained unknown. Therefore, we performed the immunohistochemical analysis of PCP4 in the following tissues: normal adrenal (NA; n=15), APA (n=15), cortisol-producing adenomas (n=15), and idiopathic hyperaldosteronism cases (IHA; n=5). APA samples (n=45) were also submitted to quantitative RT-PCR of PCP4, CYP11B1, and CYP11B2, as well as DNA sequencing for KCNJ5 mutations. Transient transfection analysis using PCP4 siRNA was also performed in H295R adrenocortical carcinoma cells, following ELISA analysis, and CYP11B2 luciferase assays were also performed after PCP4 vector transfection in order to study the regulation of PCP4 protein expression. In our findings, PCP4 immunoreactivity was predominantly detected in APA and in the zona glomerulosa of NA and IHA. In APA, the mRNA levels of PCP4 were significantly correlated with those of CYP11B2 (P<0.0001) and were significantly higher in cases with KCNJ5 mutation than WT (P=0.005). Following PCP4 vector transfection, CYP11B2 luciferase reporter activity was significantly higher than controls in the presence of angiotensin-II. Knockdown of PCP4 resulted in a significant decrease in CYP11B2 mRNA levels (P=0.012) and aldosterone production (P=0.011). Our results indicate that PCP4 is a regulator of aldosterone production in normal, hyperplastic, and neoplastic human adrenocortical cells.
Asunto(s)
Corteza Suprarrenal/metabolismo , Aldosterona/biosíntesis , Proteínas del Tejido Nervioso/metabolismo , Adenoma/genética , Adenoma/patología , Corteza Suprarrenal/efectos de los fármacos , Corteza Suprarrenal/patología , Neoplasias de la Corteza Suprarrenal/genética , Neoplasias de la Corteza Suprarrenal/patología , Angiotensina II/farmacología , Western Blotting , Línea Celular , Colforsina/farmacología , Citocromo P-450 CYP11B2/genética , Citocromo P-450 CYP11B2/metabolismo , ADN/metabolismo , Pruebas de Enzimas , Ensayo de Inmunoadsorción Enzimática , Regulación Neoplásica de la Expresión Génica , Humanos , Inmunohistoquímica , Luciferasas/metabolismo , Proteínas del Tejido Nervioso/genética , Plásmidos/metabolismo , ARN Interferente Pequeño/metabolismo , Reacción en Cadena en Tiempo Real de la Polimerasa , Esteroide 11-beta-Hidroxilasa/genética , Esteroide 11-beta-Hidroxilasa/metabolismo , TransfecciónRESUMEN
New five seven-membered vibsane-type diterpenes named 5-epi-vibsanin C, 5-epi-vibsanin H, 5-epi-vibsanin K, 18-O-methyl-5-epi-vibsanin K and 5-epi-vibsanin E have been isolated from the leaves of Viburnum awabuki (Caplifoliaceae). Their structures have been elucidated by analyses of spectroscopic data and comparison of their spectral data with those of the previously known seven-membered vibsane-type diterpenes. The occurrence of these seven-membered vibsane-type diterpenes with a cis relationship on the C-5 and C-10 positions in nature have been predicted by conformational analysis of vibsanin B, an eleven-membered vibsane-type diterpene. Vibsanin C, 5-epi-vibsanin C and 5-epi-vibsanin H exhibited moderate cytotoxic activities on KB cells.