糖皮质激素通过促使同型半胱氨酸蓄积抑制骨形成的机制研究

Since cystathionine beta-synthase (CBS) is the ?rst, and rate-limiting enzyme in the transsulfuration metabolic pathway of homocysteine (Hcy), it plays an important role in the maintenance of a normal Hcy level. In our preliminary study, we found that glucocorticoids (GCs) significantly inhibited CBS expression in differentiated osteoblasts, and increased Hcy accumulation in bone tissues. In addition, both CBS inhibitor and Hcy suppressed osteoblastic differentiation in vitro. On the basis of these results, the present proposal will firstly verify that the local Hcy accumulation does contribute to glucocorticoid-induced inhibition of osteoblastic bone formation, while intervention therapies aim at reducing local Hcy levels can attenuate glucocorticoid-induced osteoporosis. In cardiovascular and central nervous systems, it has been found that high Hcy level results in DNA hypomethylation, which subsequently leads to abnormal regulation of gene transcription, and plays critical roles in the pathogenesis of various diseases. Thus, the present project will investigate whether glucocorticoid-induced inhibition of osteoblastic bone formation is partly due to Hcy accumulation-induced DNA hypomethylation. Moreover, the direct targets and the precise promoter regions responsive to Hcy accumulation-induced hypomethylation will also be clarified. Finally, we will examine whether osteoblast-specific transcriptional factors, Runx2 and Osterix, are involved in the osteoblastic differentiation-induced CBS expression, as well as in the glucocorticoid-induced down-regulation of CBS expression. The present study will provide reliable evidence to elucidate the impact of Hcy accumulation and associated DNA hypomethylation on the pathogenesis of glucocorticoid-induced osteoporosis. Improving the impaired Hcy metabolism, or restoring the normal DNA methylation state, may become one of the future treatment options of glucocorticoid-induced osteoporosis.

同型半胱氨酸（Hcy）转硫化代谢途径的关键酶CBS对于机体Hcy正常水平的维持非常重要。我们发现糖皮质激素（GCs）可以抑制成骨细胞CBS表达，并导致骨组织中Hcy蓄积；而CBS抑制剂和Hcy均可在体外抑制成骨细胞分化。在此基础上，本项目首先将证实骨组织Hcy蓄积是GCs抑制骨形成的重要原因，而降低Hcy浓度的干预措施则能够改善糖皮质激素性骨质疏松（GIO）。鉴于Hcy蓄积已在心血管和神经系统被证实可导致基因组DNA低甲基化，本项目在明确Hcy蓄积对骨组织DNA甲基化水平影响的基础上将进而探讨其在GCs抑制成骨过程中的作用，并明确具体基因靶点。同时我们还将探讨成骨特异性转录因子Runx2和Osterix在成骨分化过程中CBS自身表达调控以及GCs抑制成骨细胞CBS表达中的作用。本项目研究将有助于阐明GIO的发生机制，并从改善Hcy代谢障碍以及表观遗传学的角度为临床治疗提供新思路。

同型半胱氨酸（Hcy）转硫化代谢途径的关键酶CBS对于机体Hcy正常水平的维持非常重要。我们发现糖皮质激素（GCs）和PPARγ激动剂罗格列酮（RSG）均可诱导骨质疏松小鼠血清和胫骨组织中Hcy含量均显著增高。此外，新生小鼠颅骨成骨样细胞加入抗坏血酸和beta-磷酸甘油进行诱导分化过程中CBS表达显著增多、并且可被地塞米松和RSG完全抑制，CSE表达则无显著变化。Hcy和 CBS阻断剂 AOAA均能显著抑制成骨细胞分化。GCs和RSG诱导骨质疏松小鼠给予外源性硫化氢（H2S）制剂则可以显著减轻骨组织Hcy蓄积，同时改善骨质疏松症状。上述结果提示GCs和RSG抑制成骨细胞CBS表达是成骨细胞内Hcy蓄积，成骨细胞分化障碍、进而导致骨质疏松的重要原因之一。GSK3β/β-catenin通路在成骨细胞分化过程中起关键作用。我们发现酪氨酸磷酸酶SHP-1可以与GSK3β结合并使其去磷酸化从而调节抑制其功能。而GCs和RSG引起的Hcy蓄积可以显著抑制SHP-1表达，从而使GSK3β酪氨酸磷酸化水平升高。激活的GSK3β进一步降解β-catenin而抑制成骨细胞分化，促进骨质疏松发生。过表达SHP-1或者阻断GSK3β信号通路均可显著逆转GCs和RSG抑制成骨分化的作用。近年来研究表明H2S可通过修饰靶蛋白半胱氨酸上的巯基形成S-硫巯基化而调节其功能，因此我们采用高通量的质谱技术深入研究了H2S改善Hcy蓄积抑制成骨分化作用的分子机制。采用质谱技术获得了可能被S-硫巯基化的靶蛋白信息后，我们发现其中F0F1-ATP合酶、VDAC1和ALDH这三种蛋白已知在线粒体功能维持中起关键作用。线粒体功能障碍已被证实在GCs诱导骨质疏松过程中起作用，同时也被报道在高Hcy血症引起的多器官功能损伤过程中起作用。那么，H2S是否确实通过对这三种线粒体蛋白巯基修饰而实现对成骨细胞功能的调节作用呢？这一潜在作用机制值得我们进行深入研究。