Abstract：
BACKGROUND: Craniofacial skeletal deformities can be addressed by applying tensile force to sutures to prompt sutural bone formation. The intricate process of mechanical modulation in craniofacial sutures involves complex biomechanical signal transduction. The small GTPase Ras homolog gene family member A (RhoA) functions as a key mechanotransduction protein, orchestrating the dynamic assembly of the cytoskeleton by activating the Rho-associated coiled-coil containing protein kinase (ROCK). Transcriptional coactivator with PDZ-binding motif (TAZ) serves as a crucial mediator in the regulation of genes and the orchestration of biological functions within the mechanotransduction signaling pathway. However, the role of RhoA/ROCK-TAZ in trans-sutural distraction osteogenesis has not been reported.
METHODS: We utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence.
RESULTS: The osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation.
CONCLUSIONS: This study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.
METHODS: We utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence.
RESULTS: The osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation.
CONCLUSIONS: This study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.
摘要：
背景：颅面骨骼畸形可以通过对缝线施加拉力以促进缝线骨形成来解决。颅面缝线中机械调制的复杂过程涉及复杂的生物力学信号转导。小GTPaseRas同源基因家族成员A（RhoA）作为关键的机械转导蛋白，通过激活Rho相关的含卷曲螺旋的蛋白激酶（ROCK）来协调细胞骨架的动态组装。具有PDZ结合基序（TAZ）的转录共激活因子在机械转导信号通路中的基因调控和生物功能编排中起着至关重要的介导剂。然而,RhoA/ROCK-TAZ在经缝牵张成骨中的作用尚未见报道。
方法：我们利用前成骨细胞特异性RhoA缺失小鼠建立了从新生小鼠中分离的前成骨细胞的体内颅骨穿缝牵张模型和体外机械拉伸模型。显微CT和组织学染色用于检测颅骨矢状缝中新骨的形成以及RhoA的激活。Osterix和TAZ。通过Westernblot检测机械张力下成骨细胞中ROCK-limk-cofilin的激活和TAZ的核易位,qRT-PCR,和免疫荧光。
结果：通过激活RhoA和Rho相关激酶（ROCK），机械张力促进了前成骨细胞的成骨分化，而RhoA的消融通过抑制缝线扩张后的成骨细胞前分化而损害成骨。此外，抑制RhoA的表达可以通过ROCK-LIM结构域激酶（LIMK）-cofilin途径阻止F-肌动蛋白的组装来阻断TAZ的拉伸刺激的核易位。此外,TAZ激动剂TM-25659可以通过增加TAZ核积累来减轻前成骨细胞中RhoA消融引起的成骨受损。
结论：这项研究表明，机械拉伸在经缝牵张成骨过程中促进前成骨细胞的成骨分化，该过程由RhoA/ROCK-TAZ信号轴介导。总的来说,我们的结果可能为通过经缝牵张成骨治疗颅骨融合症患者的潜在治疗策略提供了见解.
方法：我们利用前成骨细胞特异性RhoA缺失小鼠建立了从新生小鼠中分离的前成骨细胞的体内颅骨穿缝牵张模型和体外机械拉伸模型。显微CT和组织学染色用于检测颅骨矢状缝中新骨的形成以及RhoA的激活。Osterix和TAZ。通过Westernblot检测机械张力下成骨细胞中ROCK-limk-cofilin的激活和TAZ的核易位,qRT-PCR,和免疫荧光。
结果：通过激活RhoA和Rho相关激酶（ROCK），机械张力促进了前成骨细胞的成骨分化，而RhoA的消融通过抑制缝线扩张后的成骨细胞前分化而损害成骨。此外，抑制RhoA的表达可以通过ROCK-LIM结构域激酶（LIMK）-cofilin途径阻止F-肌动蛋白的组装来阻断TAZ的拉伸刺激的核易位。此外,TAZ激动剂TM-25659可以通过增加TAZ核积累来减轻前成骨细胞中RhoA消融引起的成骨受损。
结论：这项研究表明，机械拉伸在经缝牵张成骨过程中促进前成骨细胞的成骨分化，该过程由RhoA/ROCK-TAZ信号轴介导。总的来说,我们的结果可能为通过经缝牵张成骨治疗颅骨融合症患者的潜在治疗策略提供了见解.
