GR/Sp3/HDACs/UGDH介导孕期地塞米松暴露子代生长板发育抑制的研究

Dexamethasone is a wildly used medication during pregnancy. Recent researches indicated that prenatal dexamethasone suppressed the development of fetal growth plate, and thus resulted in a shortened body length. However, the underlying mechanisms still remains unclear. The applicant previously reported that UDP-glucose dehydrogenase (UGDH) plays a critical roles in the biosynthesis of proteoglycans (PGs) in chondrocytes. As PGs are the key inducers of chondrogenesis, the trigger of bone formation and growth, UGDH might be essential in skeleton development. Sp3 is one of the trans-inhibitors of UGDH gene, while glucocorticoid receptor (GR) is the receptor to which cortisol and other glucocorticoids bind. It’s reported that both GR and Sp3 could recruit HDAC1/2 and thus modulated the acetylation status of target histones, and that glucocorticoid could induced the interaction of GR and Sp3. Our latest process showed that both UGDH mRNA level and PGs content in the growth plate were attenuated by dexamethasone, while GR, Sp3, and HDAC2 mRNA expression were stimulated. So, we hypothesized that prenatal dexamethasone induced the interaction of GR and Sp3 and binding to UGDH’s DNA sequence, thus recruited HDAC1/2, and subsequently induced the hypo-acetylation of UGDH histone, and inhibited UGDH gene expression. Then, the suppressed UGDH gene expression led to attenuate PGs synthesis, which resulted in suppressed chondrogenesis and further caused the dysplasia of fetal growth plate. So, we believe that our study would contribute to a safer prenatal drug use and a better understanding of Dex’s developmental toxicology.

地塞米松（Dex）是妊娠期常用药物。最新发现，Dex可抑制胎儿生长板软骨发育并缩短其出生体长，但机制不明。已知UDP-葡萄糖脱氢酶（UGDH）参与调控软骨发生与发育；同时，Sp3是UGDH抑制性转录因子，糖皮质激素受体（GR）是Dex主要效应因子，两者可在皮质激素作用下结合并发挥协同作用，且GR、Sp3均可招募HDAC1/2并与之形成复合物调控靶基因组蛋白乙酰化。申请人预实验发现，Dex可抑制胎鼠生长板软骨发育，下调局部UGDH基因表达，上调GR、Sp3、HDAC1、HDAC2基因表达；同时，Dex还可抑制BMSCs成软骨分化及UGDH基因表达。因此，我们推测“Dex可能通过诱导GR、Sp3与UGDH基因结合，招募HDAC1/2，致UGDH组蛋白低乙酰化并下调其表达，进而抑制生长板发育”，并拟从动物及细胞水平予以验证，为明确Dex的骨骼系统发育毒性及作用靶标、指导临床安全用药提供依据。

本项目完成原计划内容，发现：孕9-20天地塞米松暴露(0.2 mg/kg.d)可致子代股骨及生长板长度缩短，蕃红O染色提示生长板局部蛋白多糖（PGs）含量明显降低，伴随Ugdh表达及组蛋白乙酰化水平下降；并可致胎鼠Ugdh表达及组蛋白乙酰化（H3K9ac）水平下降，伴随GR、Sp3及 Hdac1表达上调。细胞实验发现，地塞米松在大鼠BMSCs成软骨分化模型中可抑制Ugdh表达及组蛋白乙酰化水平（H3K9ac），伴随GR、 Sp3及 Hdac1表达上调；分别RU486拮抗GR、siRNA沉默Hdac1及Sp3后可逆转地塞米松对Ugdh表达及组蛋白乙酰化水平的抑制；同时，GR可与Ugdh启动子区直接结合，并分别与HDAC1、Sp3存在相互作用。这提示，地塞米松通过诱导生长板软骨细胞GR入核结合于Ugdh基因启动子区，招募HDAC1及Sp3，降低Ugdh基因启动子区乙酰化水平，抑制Ugdh基因表达，导致PGs合成减少，最终扰乱生长板发育、抑制长骨生长。.在此基础上，申请人拓展了研究内容：首先，进一步证实孕期地塞米松暴露可致子代胎鼠长骨发育不良，出生后峰值骨量降低，并存在跨代遗传效应，其机制与成骨细胞局部血管紧张素转化酶（ACE）基因组蛋白修饰改变介导的骨局部肾素-血管紧张素系统持续慢性激活有关；同时，开展了孕期咖啡因暴露所致子代长骨发育不良及成年骨质疏松易感的研究，并证实孕期咖啡因暴露可致子代胎鼠母源性糖皮质激素过暴露，并抑制长骨发育，导致出生后峰值骨量降低，而成骨细胞局部血管紧张素转化酶（ACE）基因表遗传修饰改变介导的骨局部肾素-血管紧张素系统持续慢性激活参与了上述过程；另外，申请人探讨了11β-羟基类固醇Ⅱ型脱氢酶（11β-HSD2）与孕期地塞米松暴露所致骨发育不良的相关性，并证实Hsd11b2低表达介导PDE所致的成年雄性子代骨量减少及骨质疏松症易感性增加，其发生机制为地塞米松诱导胎长骨BMSCs中GR活化并招募HDAC11入核及抑制Sp1入核，共同抑制Hsd11b2启动子区组蛋白乙酰化及表达水平，出生后Hsd11b2低功能编程改变，进而导致骨组织糖皮质激素 “屏障”破坏及成年骨量丢失，且股骨过表达Hsd11b2可逆转上述过程，提示Hsd11b2可作为胎源性骨质疏松治疗靶点。

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