Abstract：
OBJECTIVE: This study aimed to investigate the effect of endoplasmic reticulum (ER) stress sensor inositol-requiring enzyme 1α (IRE1α) on the sonic hedgehog N-terminus (N-Shh)-enhanced-osteogenic differentiation process in mouse embryonic fibroblasts (MEFs).
METHODS: Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis.
RESULTS: N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers.
CONCLUSIONS: Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.
METHODS: Osteogenesis of MEFs was observed by alkaline phosphatase (ALP) staining, alizarin red staining, and Von Kossa staining assays. Activation of unfolded protein response and Shh signaling were examined using real-time quantitative PCR and western blot assays. IRE1α-deficient MEFs were used to explore the effect of IRE1α on N-Shh-driven osteogenesis.
RESULTS: N-Shh increased ALP activity, matrix mineralization, and the expression of Alp and Col-I in MEFs under osteogenic conditions; notably, this was reversed when combined with the ER stress activator Tm treatment. Interestingly, the administration of N-Shh decreased the expression of IRE1α. Abrogation of IRE1α increased the expression of Shh pathway factors in osteogenesis-induced MEFs, contributing to the osteogenic effect of N-Shh. Moreover, IRE1α-deficient MEFs exhibited elevated levels of osteogenic markers.
CONCLUSIONS: Our findings suggest that the IRE1α-mediated unfolded protein response may alleviate the ossification of MEFs by attenuating Shh signaling. Our research has identified a strategy to inhibit excessive ossification, which may have clinical significance in preventing temporomandibular joint bony ankylosis.
摘要：
目的：本研究旨在研究内质网（ER）应激传感器要求肌醇酶1α（IRE1α）对小鼠胚胎成纤维细胞（MEFs）的声刺猬N端（N-Shh）增强成骨分化过程的影响。
方法：通过碱性磷酸酶（ALP）染色观察MEFs的成骨作用，茜素红染色，和VonKossa染色测定。使用实时定量PCR和蛋白质印迹测定检查未折叠蛋白应答的激活和Shh信号传导。使用IRE1α缺陷的MEFs来探索IRE1α对N-Shh驱动的成骨作用。
结果：N-Shh增加了ALP活性，基质矿化，以及成骨条件下MEFs中Alp和Col-I的表达；当与ER应激激活剂Tm治疗结合使用时，这种情况可以逆转。有趣的是,N-Shh的给药降低了IRE1α的表达。IRE1α的消除增加了Shh途径因子在成骨诱导的MEFs中的表达，有助于N-Shh的成骨作用。此外，IRE1α缺陷型MEFs显示成骨标志物水平升高。
结论：我们的发现表明IRE1α介导的未折叠蛋白反应可能通过减弱Shh信号来减轻MEFs的骨化。我们的研究确定了一种抑制过度骨化的策略，这可能对预防颞下颌关节骨性强直具有临床意义。
方法：通过碱性磷酸酶（ALP）染色观察MEFs的成骨作用，茜素红染色，和VonKossa染色测定。使用实时定量PCR和蛋白质印迹测定检查未折叠蛋白应答的激活和Shh信号传导。使用IRE1α缺陷的MEFs来探索IRE1α对N-Shh驱动的成骨作用。
结果：N-Shh增加了ALP活性，基质矿化，以及成骨条件下MEFs中Alp和Col-I的表达；当与ER应激激活剂Tm治疗结合使用时，这种情况可以逆转。有趣的是,N-Shh的给药降低了IRE1α的表达。IRE1α的消除增加了Shh途径因子在成骨诱导的MEFs中的表达，有助于N-Shh的成骨作用。此外，IRE1α缺陷型MEFs显示成骨标志物水平升高。
结论：我们的发现表明IRE1α介导的未折叠蛋白反应可能通过减弱Shh信号来减轻MEFs的骨化。我们的研究确定了一种抑制过度骨化的策略，这可能对预防颞下颌关节骨性强直具有临床意义。
