支架蛋白Salvador1调控Hippo-YAP/TAZ信号通路抑制软骨内成骨的作用及机制

Endochondral ossification is a key contributor to the development of the vertebral skeleton and its regulatory mechanisms are not yet clear. During endochondral ossification, Hippo pathway regulates chondrocyte differentiation at multiple steps through down-stream target effector YAP. Salvador1 (Sav1) is one of the core components of the Hippo pathway. .Research studies by our group have shown that the inhibition of Sav1 results in the limitation of the metatarsal growth, shortening of proliferative zone and a lower expression of Col10a1 in hypertrophy zone. Further studies found that knockdown Sav1 by siRNA in chondrocytes increases the expression of YAP. To date, the effect of Sav1 knockout in vivo has been demonstrated in a few specific organs, including the liver, skin and colon, not cartilage. .Therefore, we have generated cartilage-specific Sav1-knockout mice to explore the regulation of Sav1 on endochondral ossification. During the studies periods, the general observation is made at the different time point and collagen synthesis and mineralization is detected after euthanasia. By analyzing the differences between Sav1-knockout mice and control mice in Hippo-YAP/TAZ pathway and chondrogenesis-related signaling pathway, we have developed a regulatory cross talk between Sav1 and endochondral ossification, which may provide theoretical basis for the prevention and treatment of diseases related to skeletal dysplasia.

软骨内成骨是骨骼发生的主要方式，但其调控机制尚未完全明确。本团队在小鼠跖骨组织中敲除Hippo信号通路支架蛋白Salvador1（Sav1）后发现，跖骨增长受限，增殖区变短，肥大区Col10a1表达下降；小鼠软骨细胞中敲除Sav1引起下游效应分子YAP的表达增强。这说明Hippo信号通路分子Sav1在软骨细胞的增殖及分化过程中发挥重要作用。然而，Sav1对软骨内成骨及骨骼形成过程的影响尚缺乏体内数据，机理也有待进一步研究。因此，本项目将建立软骨组织中Sav1特异性敲除小鼠模型，通过分析小鼠骨骼的大体及显微结构、胶原合成及修饰、Hippo-YAP/TAZ及软骨生成相关信号通路的变化，明确Sav1敲除对软骨内成骨及骨骼形成的作用，阐明Sav1调控软骨生成及稳态的分子机制，为骨骼发育异常疾病的防治提供理论基础。

Hippo信号通路在器官发育、组织稳态等生理过程中发挥关键作用。特异性敲除Hippo信号通路上游的支架蛋白Salvador1（Sav1），会影响肝脏、肾脏、结肠的组织结构，但其对骨骼发生的作用，还未见报道。利用电转染法在跖骨组织中敲除Sav1后，跖骨增长受限。那么，Sav1是否可以作为调节软骨内成骨过程的关键分子，参与骨骼发育过程？这一问题尚待明确。.本项目利用Cre/Loxp系统在小鼠软骨细胞中，借助tamoxifen时序性调控Sav1的表达。数据显示，2周Sav1敲除鼠（cKO）的体重下降6%，其体长为对照组(Ctrl)的86%。新生第0天，Sav1cKO小鼠的体重下降10%，其体长为对照组的84%。但是在Sav1Col2alERT的体外试验中，与对照组相比，4-OHtamoxifen处理后的跖骨更长，比对照组长10%。鉴于不一致的体内外结果，本项目接下来利用Col2a1Cre工具鼠（非tamoxifen诱导型）在软骨组织中将Sav1基因敲除。令人意外的是，Sav1Col2al敲除组（cKO）中的雌鼠、雄鼠均可顺利存活。敲除组雄鼠8周生长曲线与与对照组（Ctrl）的生长曲线接近，并不存在明显差异，雌鼠也是如此。接着，本项目对4周、2周、新生Sav1Col2al敲除鼠的生长板高度进行分析，也未在增殖区及肥大区发现异常。最后，本项目对新生鼠生长板软骨细胞的凋亡、增殖及特异性蛋白表达情况进行分析，遗憾的是，敲除组和对照组的数据并无显著性差异。.综上所述，基于Col2a1Cre工具鼠（tamoxifen诱导型及非tamoxifen诱导型），本课题的工作并未发现Hippo通路上游分子支架蛋白Sav1影响软骨内成骨过程的直接证据，下一步试验的实施或将考虑纳入其他Cre工具鼠，如Prx1Cre等。