P450c17双重催化活性的差异调控在多囊卵巢综合征高雄激素血症发生中的分子机制研究

PCOS is a heterogeneous, genetically complex, endocrine disorder among women of reproductive age with prevalence of between 5 and 10%. P450c17, expressed in the adrenal and gonads, has dual catalytic activities: 17-hydroxylase activity and 17,20-lyase activity. As a branch point of cortisol and sex steroids biosynthesis, P450c17 determining the direction of steroidogenesis, is crucial in the biosynthesis of androgen. In PCOS patients, the concentrations of cortisol, reflecting 17-hydroxylase activity, remain essentially constant, whereas concentrations of DHEA rise remarkably, which suggests the potential differential regulation of two catalytic activities of P450c17. From previous study, 17,20-lyase activity can be augmented by three post-translational mechanisms: increased molar ratio of POR to P450c17, an allosteric mechanism by cytochrome b5 and the serine/threonine (Ser/Thr) phosphorylation of P450c17. .From our preliminary work, the oxidative stress model of steroidogenesis cells by treatment NCI-295R cells with hydrogen peroxide (H2O2) and palmitate. Reactive oxygen species (ROS) were observed to be increased under the treatments. By using ELISA and TLC (Thin-Layer Chromatography), 17,20-lyase activity were observed to be increased significantly but the 17-hydroxylase activity relatively stable. Pretreatment the cells with MAPK P38 inhibitor showed that the increasement of 17,20-lyase activity was alleviated. Using Western Blot, the phosphorylation of MAPK P38α was observed to increased, while the expression level of P450c17, POR and cytochrome b5 showed no significant change under oxidative stress. It is suggested that MAPK P38 activated by oxidative stress induced phosphorylation of serine of P450c17, resulting in elevation of 17,20-lyase activity..In the rat models produced by high fat diets and D-galactose injection, the ROS levels were revealed to be increased. Compared with the control group, rats treated with high fat diets and D-galactose displayed abnormal estrous cycles with increasing androgen biosynthesis. Meanwhile, multiple large cysts with diminished granulosa layers and increased thecal layers were also observed in model group..It is demonstrated that oxidative stress plays an important role in the development of IR. Based on our preliminary data, it is hypothesized that MAPK P38α activated by oxidative stress, resulting in phosphorylation of P450c17 and differential regulation of dual catalytic activities of P450c17, is crucial in the pathogenesis of hyperandrogenemia of PCOS. Future study concerning the regulation of oxidative stress to P450c17 will be performed in animal model, as well as in the isolated human thecal cells to verify the hypothesis. Phos-Tag and mass spectrometry technology will be used to detect the phosphorylated site of P450c17, gaining insight into the pathogenesis of PCOS and find new therapeutical approach.

P450c17是一种具有17-羟化酶和17,20-裂解酶双重催化活性的酶蛋白，在雄激素合成过程中起关键作用。高雄激素血症是多囊卵巢综合征（PCOS）的重要临床特征，但其分子调控通路尚不明确。课题组在NCI-295R细胞中发现，氧化应激状态下出现17-羟化酶活性相对稳定而17,20-裂解酶活性显著升高的双催化活性的差异调控，导致雄激素分泌增加，且与MAPK P38α磷酸化有关；动物模型也证实机体氧化应激状态可导致雄激素分泌升高及卵巢多囊性改变。基于上述研究，提出氧化应激是PCOS高雄激素血症和胰岛素抵抗的共同促发因素，而MAPK P38α介导P450c17磷酸化造成的双催化活性差异是导致高雄激素血症的关键调控通路。研究拟通过进一步体内外实验和人卵泡膜细胞培养对上述假说进行深入验证，进而采用Phose-Tag和蛋白质谱技术，寻找P450c17分子的磷酸化位点，为PCOS治疗开辟新的途径。

P450c17是一种具有17-羟化酶和17,20-裂解酶双重催化活性的酶蛋白，在皮质醇和雄激素合成过程中起关键作用。以往研究发现，P450c17磷酸化后会选择性增加17,20-裂解酶活性而对17α-羟化酶活性影响微弱。高雄激素血症是多囊卵巢综合征（PCOS）的重要临床特征之一，但其分子调控通路及其与胰岛素抵抗因果关系尚不明确。课题组在肾上腺NCI-295R细胞中发现，采用棕榈酸、过氧化氢（H2O2）和HNE三种氧化剂诱导氧化应激，采用ELISA, TLC 和 LC-MS/MS三种方法，检测17-羟孕酮和DHEA水平观察17α-羟化酶和17,20-裂解酶活性；发现氧化应激使P450c17酶双催化活性出现差异性调控，17-羟化酶活性相对稳定而17,20-裂解酶活性显著升高。通过Western blot检测调控P450c17活性的三种机制中的重要分子——POR、 b5和P450c17，发现氧化应激下三种分子水平相对稳定，而应激感受分子MAPK p38α磷酸化显著增强。我们进一步采用p38α抑制剂（SB203580和B202190）干预和RNA干扰，发现抑制或下调p38α几乎逆转了棕榈酸（0.45-0.75mM）和H2O2引起的DHEA水平升高，而17-羟孕酮水平仍然保持相对稳定。上述的研究结果提示氧化应激是PCOS高雄激素血症和胰岛素抵抗的共同促发因素，而MAPK P38α介导P450c17磷酸化造成的双催化活性差异是导致高雄激素血症的关键调控通路。相关结果已发表在Mol Cell Endocrinol。.在基金的资助下，共发表SCI论文8篇，包括：1.建立靶向二代测序技术对70例46，XY 性发育异常患者进行致病基因分布研究；2. 对15例中国人17-羟化酶缺陷进行了临床和突变特点研究；3.对中国人第1例CYP19A1突变的导致46，XX DSD进行了的临床和分子功能研究；4.描述14例5α还原酶缺陷症患者，并对3个新的SRD5A2突变进行功能研究； 5. 对LHR新的剪切异构体的功能及其剪切位点SNP与无精症的相关性进行了研究；6. 对第1例IVF生育的雄激素不敏感综合征进行临床特点和基因突变研究；7.对甲减导致的急性肾损伤进行的临床研究。共发表中文核心论著2篇综述2篇，包括对33例成人单纯男性化型21-羟化酶缺陷临床特点和基因型研究等。