Rennert H, Fischer R T, Alvarez J G, Trzaskos J M, Strauss J F
Department of Obstetrics and Gynecology, University of Pennsylvania, School of Medicine, Philadelphia 19104.
Endocrinology. 1990 Aug;127(2):738-46. doi: 10.1210/endo-127-2-738.
De novo synthesis of cholesterol and low-density lipoprotein (LDL) receptor levels are suppressed in the presence of cholesterol. Recent evidence suggests that a cholesterol metabolite (possibly a hydroxysterol), not cholesterol per se, is the effector that inhibits transcription of genes encoding enzymes involved in sterol synthesis and LDL receptors. We found that 26-hydroxycholesterol inhibits human ovarian cell sterol synthesis, and that luteinized human granulosa cells contain 26-hydroxylase messenger RNA (mRNA). We proceeded to characterize the enzyme generating 26-hydroxycholesterol in the rat ovary. Mitochondria derived from ovaries of PMSG-human CG (hCG) primed immature rats (day 3 post-hCG) metabolized [3H] cholesterol into [3H]26-hydroxycholesterol in the presence of nicotinamide adenine dinucleotide phosphate and aminoglutethimide (100 micrograms/ml), added to inhibit metabolism of sterols by the cholesterol side-chain cleavage system. The identity of the product was confirmed by chromatography in several systems; recrystallization to constant specific activity and mass spectrometry. Negligible 26-hydroxylase activity was detected in other ovarian subcellular fractions. 26-Hydroxycholesterol formation progressed at a linear rate for up to 40 min and was linearly related to mitochondrial protein added to the incubation mixture. 26-Hydroxylase was markedly stimulated (5-fold) by calcium (0.2 mM). Maximal rates of 26-hydroxycholesterol formation observed were 1 pmol/min.mg protein. This activity is substantially lower than cholesterol side-chain cleavage measured in the absence of aminoglutethimide. Ketoconazole (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner. Pregnenolone (1-1000 microM) and progesterone (1-100 microM) inhibited 26-hydroxylase in a dose-dependent manner, with appreciable inhibitory effects in the 1-10 microM range. We suggest that 26-hydroxycholesterol is an intracrine regulator that controls cellular sterol metabolism. Formation of 26-hydroxcholesterol in ovarian cells may be regulated by steroidogenic activity in such a way as to ensure availability of steroid hormone precursors. When steroidogenesis is active, 26-hydroxylase is inhibited by products of the side-chain cleavage system, allowing increased de novo sterol synthesis and LDL uptake. With reduced steroidogenic activity and less demand for cholesterol, 26-hydroxylase is not blocked, permitting formation of 26-hydroxycholesterol with attendant reduction in sterol synthesis and LDL receptor gene expression.
在有胆固醇存在的情况下，胆固醇的从头合成及低密度脂蛋白（LDL）受体水平会受到抑制。最近的证据表明，抑制参与甾醇合成的酶及LDL受体编码基因转录的效应物是一种胆固醇代谢产物（可能是一种羟基甾醇），而非胆固醇本身。我们发现26-羟基胆固醇可抑制人卵巢细胞的甾醇合成，且黄体化的人颗粒细胞含有26-羟化酶信使核糖核酸（mRNA）。我们进而对大鼠卵巢中生成26-羟基胆固醇的酶进行了特性分析。从经孕马血清促性腺激素-人绒毛膜促性腺激素（hCG）预处理的未成熟大鼠（hCG注射后第3天）卵巢中分离得到的线粒体，在添加烟酰胺腺嘌呤二核苷酸磷酸及氨鲁米特（100微克/毫升）以抑制胆固醇侧链裂解系统对甾醇的代谢作用后，可将[3H]胆固醇代谢为[3H]26-羟基胆固醇。产物的同一性在多个系统中通过色谱法得以证实；通过重结晶使其比活性恒定并进行了质谱分析。在其他卵巢亚细胞组分中检测到的26-羟化酶活性可忽略不计。26-羟基胆固醇的形成在长达40分钟内呈线性速率进行，且与加入孵育混合物中的线粒体蛋白呈线性相关。26-羟化酶受到钙（0.2毫摩尔）的显著刺激（5倍）。观察到的26-羟基胆固醇最大生成速率为1皮摩尔/分钟·毫克蛋白。该活性显著低于在无氨鲁米特时测得的胆固醇侧链裂解活性。酮康唑（1 - 100微摩尔）以剂量依赖方式抑制26-羟化酶。孕烯醇酮（1 - 1000微摩尔）和孕酮（1 - 100微摩尔）以剂量依赖方式抑制26-羟化酶，在1 - 10微摩尔范围内有明显的抑制作用。我们认为26-羟基胆固醇是一种控制细胞甾醇代谢的自分泌调节因子。卵巢细胞中26-羟基胆固醇的形成可能受类固醇生成活性的调节，以确保类固醇激素前体的可利用性。当类固醇生成活跃时，26-羟化酶受到侧链裂解系统产物的抑制，从而使甾醇的从头合成及LDL摄取增加。随着类固醇生成活性降低且对胆固醇的需求减少，26-羟化酶未被阻断，从而允许26-羟基胆固醇的形成，随之甾醇合成及LDL受体基因表达减少。
