Rac1调控Sonic Hedgehog信号的分子机制及意义研究

Vertebrate bone development requires precise regulations from several conserved inductive signals, such as Sonic Hedgehog(Shh) signal. Plenty of evidence in clinical has demonstrated that Shh signal loss or mutation will cause severe abnormilities in bone development. However, the mechanisms are poorly understood. In our long-term exploration of Shh signal transduction, we found that Shh signal is regulated by Rac1. Rac1 knockdown or inhibition of Rac1 activity gives rise to activation of Shh signal, whereas overexpression of continuously activated Rac1(caRac1) inhibits Shh signal. In addition, activation of Shh signal restrains Rac1 activity. Therefore, we speculate that Rac1 is involved in Shh siganl transduction. Based on our premier data, we will further study and clarify the detailed mechanism(s) of Rac1 effects in Shh signal, and the potential role(s) of Rac1 in Shh-directed limb bud patterning. By this study, we will have a deeper understanding and further revealing of Shh signal and Rac1 function,and provide with the theoretical basis and effective strategies for the prevention and treatment of skeletal development-related diseases.

脊椎动物的骨发育受多条保守信号的精确调控，如Sonic Hedgehog（Shh）信号。大量的临床证据表明该信号的缺失或突变会导致骨发育的严重异常，但其致病分子机制不详。在长期探索Shh信号调控机制的研究过程中，我们发现Rac1显著影响Shh信号的活性：敲降Rac1或抑制Rac1活性激活Shh信号，而过表达激活形式的Rac1(caRac1)则显著抑制Shh信号；并且，Shh信号的激活亦能抑制Rac1活性。因此我们推测，Shh信号通过影响Rac1活性从而调控信号的下游事件。以上述结果为基础，本课题将利用体外细胞系模型以及基因修饰的小鼠模型，进一步研究并阐明Rac1调控Shh信号的具体效应和分子机制，以及可能的在肢芽早期发育中的重要生理意义。本课题不仅揭示Shh信号调控的新机制，而且进一步充实Rac1的的生理效应；也将为骨发育调控提供新的理论，为临床相关疾病的防治提供靶点和参考。

背景：转录因子Gli的核转位对Hedgheog（Hh）信号的活性至关重要，Hh的异常激活会引起肿瘤的发生发展。然而，Gli核转位的分子机制仍知之甚少。在本研究中，我们发现Hh信号引起的Gli核转位依赖Rac1激活。.方法：我们利用C3H10T1/2细胞系和分离的小鼠原代成纤维细胞研究Rac1活性调控Hh信号的分子机制。此外，我们利用Hh激活和Rac1敲除的转基因小鼠以及人髓母细胞瘤组织样本观察Rac1在Hh介导的胚胎期肢芽发育和髓母细胞瘤进展中的作用。.结果：我们发现，Hh与受体Ptch1的结合解除了Ptch1对受体蛋白Smo的抑制效应，活化了的Smo与Vav2结合并磷酸化Vav2的Y172位点从而激活Rac1。同时，活化了的Rac1上调下游效应分子PAK1的磷酸化水平从而进一步促进KIF3A的S689和T694位点磷酸化；磷酸化的KIF3A与IFT88结合增强其蛋白稳定性而促使Gli从与SuFu形成的复合体中解聚出来，最终入核激活下游靶基因的转录。另一方面，我们证实在Smo激活引起的小鼠脑胶质瘤组织和人Shh型髓母细胞瘤组织中，Vav2的Y172位点磷酸化水平显著上调，而在小鼠肢芽条件性敲除Rac1基因则明显抑制了Gli的核转位和Hh信号活性。.结论：因此，我们的结果揭示了Hh激活诱导Gli核转位的新机理，为由该信号异常激活引起的疾病的防治提供了新的理论依据和参考。