Slominski Andrzej T, Zmijewski Michal A, Semak Igor, Sweatman Trevor, Janjetovic Zorica, Li Wei, Zjawiony Jordan K, Tuckey Robert C
Department of Pathology, University of Tennessee Health Science Center, Memphis, Tennessee, United States of America.
PLoS One. 2009;4(2):e4309. doi: 10.1371/journal.pone.0004309. Epub 2009 Feb 3.
Since P450scc transforms 7-dehydrocholesterol (7DHC) to 7-dehydropregnenolone (7DHP) in vitro, we investigated sequential 7DHC metabolism by adrenal glands ex vivo. There was a rapid, time- and dose-dependent metabolism of 7DHC by adrenals from rats, pigs, rabbits and dogs with production of more polar 5,7-dienes as detected by RP-HPLC. Based on retention time (RT), UV spectra and mass spectrometry, we identified the major products common to all tested species as 7DHP, 22-hydroxy-7DHC and 20,22-dihydroxy-7DHC. The involvement of P450scc in adrenal metabolic transformation was confirmed by the inhibition of this process by DL-aminoglutethimide. The metabolism of 7DHC with subsequent production of 7DHP was stimulated by forscolin indicating involvement of cAMP dependent pathways. Additional minor products of 7DHC metabolism that were more polar than 7DHP were identified as 17-hydroxy-7DHP (in pig adrenals but not those of rats) and as pregna-4,7-diene-3,20-dione (7-dehydroprogesterone). Both products represented the major identifiable products of 7DHP metabolism in adrenal glands. Studies with purified enzymes show that StAR protein likely transports 7DHC to the inner mitochondrial membrane, that 7DHC can compete effectively with cholesterol for the substrate binding site on P450scc and that the catalytic efficiency of 3betaHSD for 7DHP (V(m)/K(m)) is 40% of that for pregnenolone. Skin mitochondria are capable of transforming 7DHC to 7DHP and the 7DHP is metabolized further by skin extracts. Finally, 7DHP, its photoderivative 20-oxopregnacalciferol, and pregnenolone exhibited biological activity in skin cells including inhibition of proliferation of epidermal keratinocytes and melanocytes, and melanoma cells. These findings define a novel steroidogenic pathway: 7DHC-->22(OH)7DHC-->20,22(OH)(2)7DHC-->7DHP, with potential further metabolism of 7DHP mediated by 3betaHSD or CYP17, depending on mammalian species. The 5-7 dienal intermediates of the pathway can be a source of biologically active vitamin D3 derivatives after delivery to or production in the skin, an organ intermittently exposed to solar radiation.
由于细胞色素P450胆固醇侧链裂解酶（P450scc）在体外可将7-脱氢胆固醇（7DHC）转化为7-脱氢孕烯醇酮（7DHP），我们对肾上腺离体时7DHC的后续代谢进行了研究。大鼠、猪、兔和犬的肾上腺对7DHC进行了快速、时间和剂量依赖性的代谢，通过反相高效液相色谱法（RP-HPLC）检测到产生了极性更强的5,7-二烯。根据保留时间（RT）、紫外光谱和质谱，我们确定了所有受试物种共有的主要产物为7DHP、22-羟基-7DHC和20,22-二羟基-7DHC。DL-氨基谷氨酰胺对这一过程的抑制作用证实了P450scc参与肾上腺的代谢转化。毛喉素刺激了7DHC的代谢并随后产生7DHP，表明环磷酸腺苷（cAMP）依赖性途径参与其中。7DHC代谢产生的比7DHP极性更强的其他次要产物被鉴定为17-羟基-7DHP（在猪肾上腺中存在，但大鼠肾上腺中不存在）和孕-4,7-二烯-3,20-二酮（7-脱氢孕酮）。这两种产物均代表肾上腺中7DHP代谢的主要可鉴定产物。对纯化酶的研究表明，类固醇急性调节蛋白（StAR）可能将7DHC转运至线粒体内膜，7DHC可与胆固醇有效竞争P450scc上的底物结合位点，3β-羟基类固醇脱氢酶（3βHSD）对7DHP的催化效率（V(m)/K(m)）是对孕烯醇酮催化效率的40%。皮肤线粒体能够将7DHC转化为7DHP，并且7DHP会被皮肤提取物进一步代谢。最后，7DHP、其光衍生物20-氧代孕烷骨化醇和孕烯醇酮在皮肤细胞中表现出生物活性，包括抑制表皮角质形成细胞、黑素细胞和黑色素瘤细胞的增殖。这些发现定义了一条新的类固醇生成途径：7DHC→22(OH)7DHC→20,22(OH)(2)7DHC→7DHP，根据哺乳动物物种的不同，7DHP可能会由3βHSD或细胞色素P450 17α-羟化酶（CYP17）介导进行进一步代谢。该途径中的5-7二烯中间体在输送至皮肤或在皮肤中产生后，可能成为具有生物活性的维生素D3衍生物的来源，皮肤是一个间歇性暴露于太阳辐射的器官。
